Cited2 [CBP/p300-interacting transactivators with glutamic acid (E) and aspartic acid (D)-rich tail 2] is one of the founding members of a new family of transcriptional modulators. My laboratory cloned Cited2 and showed that Cited2 is induced by many cytokines and it is a transforming gene when overexpressed. We generated Cited2 null mice and found that deletion of Cited2 causes embryos to die at mid- to late gestation with several developmental defects. Since Cited2 expression correlates positively with LTR-HSCs activity and induces the expression of Bmi-1, a polycomb group gene necessary for the maintenance and renewal of embryonic and adult stem cells, we studied the role of Cited2 in fetal liver hematopoiesis. Cited2-/- fetal liver displayed significant reduction in the numbers of hematopoietic cells in different lineages. In vitro CFU and in vivo reconstitution studies demonstrate that Cited2 plays an important role in fetal liver hematopoiesis. Consistently, microarray analysis and real-time RT-PCR analyses identified several differentially expressed genes corresponding to HSC and progenitor dysfunction in Cited2-/- fetal liver. We have shown that Cited2 is a negative regulator of HIF-1 in certain tissues during development. More recently, others showed that FoxO3 is activated under hypoxia and inhibits HIF-1-induced apoptosis through Cited2 and decreased expression of Cited2 was found in FoxO triple knockout mice, establishing Cited2 as a downstream target of FoxO transcription factors. Since regional hypoxia in the bone marrow plays an important role in regulating stem cell function and FoxOs are critical mediators of HSC resistance to oxidative stress and maintenance of the HSC pool, we would like to study the crosstalk between Cited2, HIF-1, FoxOs and Bmi-1 in adult hematopoiesis using conditional knockout approach to test the hypothesis that the FoxO/Cited2/HIF-1 pathway and Bmi-1 play an important role in regulating HSC functions. The overall strategy of the application is to first functionally characterize Cited2 knockout phenotypes in adult hematopoietic tissue (Aim 1). This will be followed by two mechanistic studies to understand Cited2 actions. In addition to focusing on specific molecules such as Bmi-1 (Aim 2), FoxO3/HIF-1 (Aim 3) and their associated pathways, a more genome-wide approach will also be undertaken in parallel to uncover novel mechanisms. The proposed studies therefore will advance our knowledge of molecular mechanisms regulating normal and abnormal HSC biology, and thus may offer new insights into the therapeutic development targeting HSC for regenerative medicine and for treatment of different hematological malignancies. PUBLIC HEALTH RELEVANCE: This proposal will not only provide information critical to our understanding of normal hematopoiesis and stem cell biology but also elucidate the complex interplay between transcription factors/cofactors and signaling molecules in vivo, which may provide new clues to the molecular pathogenesis of leukemogenesis and help identify targets for specific therapies.